POPULATION ASSAY: PROAMP - Mesa Labs

POPULATION ASSAY:PROAMPLOT#: _______________________Fill Volume: ________ mlORGANISM: _______________________________POPULATION LEVEL: ______________________Performed By: _____________________________________Date: ______________________PROCEDURE:1.0 Aseptically wipe down 5 ProAmps with 70% Isopropyl alcohol and place in sterile 50mL conical tube.Using a sterile pestle or other suitable device, aseptically crush the ampoules. Aseptically withdraw 1.0mLof the pooled solution and place into a sterile screw-capped 10mL test tube containing 9.0mL of sterile,processed water.2.0 Heat shock in a water bath (10 minutes at 80º-85ºC for mesophiles and 15 minutes at 95º - 100ºC forthermophiles). Immediately cool in a water bath of 0º - 4ºC.Start Time/Temperature:_____________________/_________ºCEnd Time:___________________3.0 Vortex the tube for 15-20 seconds.4.0 Perform serial dilutions by pipetting out 1.0 mL of the aliquot into another sterile, screw-capped 10 mL testtube containing 9.0 mL of sterile, processed water. Repeat from step 3 until desired dilution factor isreached.5.0 At the dilution expected to yield 10-300 CFU, pipette out 1.0mL into each of three Petri plates. Repeat forthe final dilutions.6.0 Within 20 minutes, add approximately 20 mL TSA, pre-sterilized and cooled to 47º + 2ºC. Swirl todistribute spores evenly in agar and allow to solidify.TSA Lot # _________________________TSA Temperature:________________ºC7.0 Invert and incubate the plates (30º-35ºC for mesophiles, 55º-60ºC for thermophiles).Incubation Start Time:______________________Incubator #:______________8.0 Examine all plates at 24 (+1) hours. Record on the back the number of colony forming units(CFU's) per plate. Record the average on the following page.9.0 Calculate the average number of CFUs/ampoule from the above data using the formula on the followingpage.Performed By:___________________________________________Date:____________________________LWI-1519 Rev 2 Effective: March 15, 2010Raven Labs Page 1 of 2Omaha, NE

POPULATION ASSAY:PROAMPTotal @ 24 hrs / number of plates counted x DF x AVF = CFU/ampouleDF= Dilution factor (absolute value of the reciprocal of the dilution)AV= Average number of colonies per ampouleAFV=Ampoule fill volumeIncubation End Time: __________________________# dilutions __________CFU COUNTS AT 24 HOURSPlates 1._____________ 2._____________ 3._____________Total @ 24hours: ______________________Total @ 24 hrs__________ / 3 x _________(DF) x ________(AFV) =______________(AV)CFU/ampoule__________________________________________________________________________________________CFU COUNTS AT 24 HOURS# dilutions __________Plates 1.____________ 2._____________ 3.______________Total @ 24 hours: ____________________Total @ 24 hrs__________ / 3 x __________(DF) x ________(AFV)=______________(AV)CFU/ampoule# of Dilutions = Dilution Factor1 = 102 = 1003 = 10004 = 100005 = 1000006 = 1000000Sum of the AV of both dilutions / 2 =CFU/ ml_________________ / 2 =_____________________________________x10CFU/ampouleRead By:_________________________________________________Date:____________________LWI-1519 Rev 2 Effective: March 15, 2010Raven Labs Page 2 of 2Omaha, NE